Note: Descriptions are shown in the official language in which they were submitted.
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ISOLATION OF TETRACYCLINE DERIVATIVES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing
date of United States Provisional Patent Application No.
60/792,814 filed April 17, 2006, the disclosure of which is
hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a process for the
isolation of nitrated tetracycline derivatives.
[0003] Tetracyclines are a group of broad-spectrum
antibiotics that contain four hydrocarbon rings. Tetracycline
itself was first described in J. Am.. Chem. Soc., 1953, 75,
4621. Various tetracycline derivatives have been described
since, examples of which are described in U.S. 2,980,584;
U.S. 2,990,331; U.S. 3,062,717; U.S. 3,165,531; U.S.
3,454,697; U.S. 3,557,280; U.S. 3,674,859; U.S. 3,957,980;
U.S. 4,018,889; U.S. 4,024,272; and U.S. 4,126,680.
R" R R N
H =
7 OH
4 3
Z NHZ
9 I ~ 11 ~ - ~ ~
12 =
OH
ON 0 OH 0 0
[0004] Tetracycline derivatives are chemically stable in
strong mineral acids, particularly in concentrated sulfuric
acid, and, therefore, can be subjected to certain chemical
transformations (J. Am. Chem. Soc., 1960, 82, 1253, J. Med.
Chem., 1962, 5(3), 538). Extensive research has been performed
on nitration and halogenation of tetracyclines that afforded
7- and/or 9-halo/nitro derivatives.
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[0005] An effective method of isolating a product from the
nitration reaction mixture is precipitation with diethyl ether
(GB 876,500; EP 535346; Us 5,248,797; US 5,281,628;
US 5,401,863). Although ethers are suitable solvents for
precipitation of organic salts, diethyl ether being the most
widely used for this purpose, such solvents are inconvenient
for industrial processes because of related safety issues.
[0006] Isolation of minocycline using diethyl ether,
according to the prior art, afforded a hardly filterable and
very hygroscopic solid, even after washing it on a filter
several times with ether. Although suspending the initially
isolated product in another portion of ether afforded easily
processable non-hygroscopic material, the total amount of
ether used for finally isolating the product in a stable dry
form was very large.
[0007] An alternative isolation process is described in
J. Med. Chem., 1962, 5(3), 538, and is performed by diluting
the reaction mixture with water followed by extraction with
1-butanol. The desired solid product is then isolated from the
organic solution by means of evaporation to dryness. This
alternative process is limited to nitrated tetracycline
derivatives that are soluble in 1-butanol and is applicable to
a limited number of tetracycline derivatives.
[0008] WO 2006/130501 discloses, such as in comparative
Example 2 and Example 2 thereof, yet another method wherein
isopropanol or mixtures of isopropanol and heptane are used to
quench the reaction and then additional heptane is added to
obtain tigecycline having a suitable purity.
{0009] However, there is a need for alternative methods for
the isolation of tetracycline derivatives.
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SUMMARY OF THE INVENTION
[0010] The present invention provides a process = for
isolating nitrated tetracycline derivatives from a reaction
mixture, which is the result of the nitration of tetracycline
derivatives, by precipitation using a C3-C8 alcohol without the
need for an additional antisolvent. Preferably, the alcohol
used is a C3-C4 alcohol, even more preferably the alcohol is
isopropanol.
DETAILED DESCRIPTION
[0011] The present invention provides a process for
isolating nitrated tetracycline derivatives from a reaction
mixture, which is the result of the nitration of tetracycline
derivatives, by precipitation using only a C3-C8 alcohol.
[0012] In comparison with the prior art, it has been
surprisingly found that isopropanol alone is sufficient to
precipitatetetracycline derivatives and that the resulting
tetracycline derivatives have a chromatographic purity of
greater than about 60%, preferably having a chromatographic
purity greater than about 77%.
[0013] Moreover, it has been found that isolation of the
nitrated tetracycline derivatives allows for a dry non-
hygroscopic nitration product to be easily obtained.
[0014] This process comprises providing a reaction mixture,
which is the result of nitration of a tetracycline derivative,
combining the reaction mixture with a C3-C8 alcohol to obtain a
precipitate, and recovering the nitrated tetracycline
derivatives.
[0015] Nitration of the tetracycline derivative may be
performed by any method known in the art, such as described in
J. Med. Chem., 1994, 37, 184; US 5,248,797, US 5,401,863.
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[0016] The nitrating agent used for the nitration of the
tetracycline derivative includes, but is not limited to,
nitric acid, nitr'ic oxide or metal nitrates. The nitrating
agent may be added portionwise to the solution of tetracycline
derivative in concentrated or pure acid. This procedure may
help in obtaining a higher purity of the product.
[0017] The reaction mixture obtained following the
nitration reaction may contain a pure acid or concentrated
acid solution such as sulfuric acid, methanesulfonic acid,
trifluoroacetic acid, trifluoromethanesulfonic acid (triflic
acid), phosphoric acid, or perchloric acid.
[0018] The reaction mixture may contain, in addition to the
tetracycline derivative, nitric acid in concentrated sulfuric
acid, metal nitrate in concentrated sulfuric, or nitric acid
in acetic anhydride.
[0019] In a preferred embodiment of the present invention,
the C3-C8 alcohol is a, C3-C4 alcohol, most preferably
isopropanol. Preferably, the tetracycline derivative (weight)
to alcohol (volume) ratio ranges from about 1:20 to about
1:120, more preferably from about 1:35 to about 1:90, and most
preferably from about 1:50 to about 1:80. Preferably, the
sulfuric acid to alcohol (volume) ratio is from about 1:2.5 to
about 1:30, more preferably from about 1:4 to about 1:25, and
most preferably from about 1:6 to about 1:20.
[0020] In a preferred embodiment of the present invention,
the tetracycline derivative is selected from
6-demethyltetracycline, bromotetracycline, chlorotetracycline,
clomocycline, demeclocycline, doxycycline, lymecycline,
meclocycline, methacycline, minocycline, oxytetracycline,
rolitetracycline, tetracycline, sancycline, 5a,6-
anhydrotetracycline, DDA-tetracycline, dactylocyclinone and
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8-methoxychlorotetracycline. The tetracycline derivative also
includes the optical isomers of the compounds mentioned above.
More preferably, the tetracycline derivative is selected from
doxycycline, methacycline, sancycline and minocycline. Even
more preferably, the tetracycline derivative is selected from
7-halosancycline, 9-halosancycline and minocycline. Most
preferably, the tetracycline derivative is minocycline.
[0021] In a preferred embodiment of the present invention,
the C3-C8 alcohol is introduced in small portions into the
reaction mixture until a precipitate appears, whereupon the
remainder of the C3-C8 alcohol is introduced into the mixture.
Most preferably, the alcohol is initially added dropwise to
the reaction mixture. This method is preferred as the
subsequent filtration of the reaction mixture is faster than
known procedures, i.e. pouring the reaction mixture into the
organic solvent. Moreover, this procedure enables one to avoid
the exothermic heating of the reaction mixture as a result of
the addition of an alcohol to a concentrated acid. Indeed,
the exothermic nature of the reaction may reduce the purity of
the final product.
[0022] Introduction of the C3-C8 alcohol may be followed by
stirring the resulting reaction mixture for about 5 to about
15 hours.
[0023] In a preferred embodiment of the present invention,
an inert gas is bubbled through the reaction mixture just
before and during the addition of the alcohol to the reaction
mixture. This procedure increases the product quality by
removing nitrogen oxides which may be formed as a result of
local heating. The inert gas is preferably nitrogen or argon,
most preferably nitrogen.
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[0024] In a prefered embodiment of the present invention,
the nitrated tetracycline derivative is recovered by any
method known in the art, such as filtration and drying of the
obtained product. Preferably, the filtration temperature is
from about 0 C to about 40 C, more preferably from about ZO C
to about 30 C, and most preferably from about 20 C to about
25 C.
[0025] In a most preferred embodiment of the present
invention, filtration of the precipitate obtained after the
organic solvent is combined is performed in a nitrogen stream
so that the wet solid would not be exposed to air. Hence, when
the solid nitration product becomes a pourable powder, it can
be stored without special precautions. The obtained product
can be dried in an oven, if necessary. Filtration in an inert
gas stream also reduces the amount of the alcohol used for
washings of the nitrated tetracycline derivative and results
in initially dried solid product which is already insensitive
to moisture.
[0026] Having described the invention with reference to
certain preferred embodiments, other embodiments will become
apparent to one skilled in the art from consideration of the
specification. The invention is further defined by reference
to the following examples describing in detail the analysis of
the tetracycline derivatives and methods for preparing the
tetracycline derivatives of the invention. It will be apparent
to those skilled in the art that many modifications, both to
materials and methods, may be practiced without departing from
the scope of the invention.
[0027] EXAMPLES
[0028] Nitration of minocycline and isolation of crude
9-nitrominocycline
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[0029] Example 1 -
N N N
H H - H N =
=_ - OH ~ a ? OH
KN0y
NHZ H2SO4 98% NH2
O zN =
OH OH
OH 0 OH 0 0 OH 0 OH 0 0
Minocycline hydrochloride (5g) was completely dissolved in
H2SO9 98% (30m1), whereupon the mixture was cooled to about
0 C-5 C and solid KN03 (1.22g, 1.2eq.) was charged into the
solution. When the reaction was completed (as determined by
HPLC), cold iso-propanol was added dropwise, under cooling at
0 C-5 C, until a precipitate started to form. The remaining
solvent was run continuously into the suspension (total amount
of IPA was about 380m1). The resulting suspension was stirred
overnight and filtered under nitrogen in such manner that when
all the filtrate was passed through the funnel, the vacuum was
disconnected and the solid was further dried in the nitrogen
stream. If suitable equipment is available, the filtration can
alternatively be performed under nitrogen pressure, i.e.
similarly to a common industrial method of filtration. The
initially dried material was further dried in a vacuum oven at
about 40 C overnight to afford the desired product as a yellow
to brownish solid stable under regular conditions.
[0030] Example 2 -
Minocycline hydrochloride (60gr) was completely dissolved in
H2SO4 98% (360ml), whereupon the mixture was cooled down about
0 C-5 C and solid KNO3 (14.1gr) was introduced into the
solution portion-wise during 1.5-2 hours. When the reaction
was complete (based on HPLC), cold iso-propanol was added
dropwise, under cooling at 0 C-5 C, until a precipitate
started to form. The remaining solvent was then run
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continuously into the suspension (total amount of IPA is
3000m1). The resulting suspension was stirred for 1 hour and
filtered under nitrogen in such manner that when all the
filtrate was passed through the funnel, the vacuum was
disconnected and the solid was further dried in the nitrogen
stream. The initially dried material was further dried in a
vacuum oven at about 40 C overnight to afford 79.4gr of the
desired product, which was characterized by chromatographic
purity of about 70.6%.
[0031] Example 3 -
Minocycline hydrochloride (5gr) was completely dissolved in
HZSOq 98% (30ml), whereupon the mixture was cooled down to
about 0 C -5 C and solid KN03 (1.22gr) was introduced into the
solution. When the reaction was complete, (based on HPLC,)
cold iso-propanol was added dropwise, under cooling at
0 C-5 C, until a precipitate started to form. The remaining
solvent was run continuously into the suspension (total amount
of IPA is 375ml). The resulting suspension was stirred
overnight at ambient temperature, and then filtered and washed
twice with about 20mL portions of IPA under nitrogen. When
all the filtrate was passed through the funnel, the vacuum was
disconnected and the solid was further dried in the nitrogen
stream. The initially dried material was further dried in a
vacuum oven at about 40 C overnight to afford 7.77gr of the
desired product, which was characterized by chromatographic
purity of about 77%.
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